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Basic Papers

Terms in this set (20)

What are the four main opsins?
Channelrhodopsin: ion channel permeable to +ve ions, lead to excitation.
Halorhodopsin: pumps chloride ions in when active, lead to hyperpolarisation.
Light-driven proton pump: pumps protons out when illuminated, hyperpolarising.
Microbial type I: 7 transmembrane domains, transretinal reaction on activation allows ions across (i assume depolarisation).
Which of the four main opsin classes does archaerhodopsin fall into? Describe its role.
Light-driven proton pump. Arch-3 is commonly used, it is activated by green or yellow light and pumps positive ions out, leading to hyperpolarisation.
What is ArchT?
ArchT is an imporvement of Arch, and is 3.5x more light sensitive.
How might we express two different opsins in the same cell?
E.g. Channerhodopsin and halorhodopsin linked by a ribosomal skip peptide allows for depol by blue light and hyperpol by yellow light.
Lima, Misenbock et al., (2005) - P2X2R
Drosophila, purinoreceptors: P2X2 expressed in giant fibre cells, using GAL4-UAS transgene system. GF cells involved in escape behaviour, and when using photolysis of caged ATP. Escape behaviour shown on uncaging. Purinoreceptors not present in drosophila, but are in many mammals.
What is the general irradiances required to activate channelrhodopsins?
0.1-10mW/mm2, so background light does not typically interfere.
Do all organisms have all-trans-retinal? What problems does this pose?
High enough levels in mammals, so they don't need supplementation, but C.elegans, Drosophila etc need dietary supplementation.
Why might we not use the rat opsin, or the mammalian melanopsin?
They have slower kinetics to channelrhodopsins etc.
What are the major caveats to using rTRPV1 in mammals?
Mammalian systems operate over narrower temperatures. It is usually activated at noxiously high temps e.g. 42 degrees C, which cannot reasonably be achieved with ambient warming without affecting animal activity or harming cells.
Huang et al., (2010): Nanoparticles
TRPV1 receptors respond to nanoparticles heated suing RF magnetic field. In solution, heating of nanoparticle was measured but no solution bulk heating. Tested in human embryonic kidney cells which express calcium sensor (troponin XL) and TRPV1 - showed increase in EC calcium. Voltage sensitive dye in hippocampal neurones. C. elegans showed thermal avoidance, they proved it was particles by incubating them with fluorescent nanoparticles, and when the MF was applied, the intensity of fluorescence decreased indicating heating of C. elegans amphid area.
Give one way in which we genetically target specific cell types.
by mimicking endogenous gene expression. The simplest and most widely used method is to isolate cis-regulatory elements (enhancers and promoters) that specify such expression, and use these elements to drive the cDNA that encodes the desired protein as a transgene. Downside? Cis reg elements are often located tens or even hundreds of kilobases away from the gene they regulate, making it difficult to generate conventional transgenics that include all relevant elements. In addition, because the transgene integrates randomly in the genome, its expression will be influenced by local regulatory elements.
Struber et al., (2011): Amygdala and Reward Seeking
BLA -> NAc neurones in mice. AAV delivery w/ ChR2 w/yfp. Nosepokes measured b/w two flaps. Nosepokes increased when stimulating these neurones. D1R antagonist abolished effect, D2R had no effect. Rate of responding of mice did not change overall. Light gated Cl- channel injeted via virus in BLA, sucrose drink task. Licks measured of two solutions. Attenuation of licking in response to cue or subsequent delivery. Also reduced number of licks throughout the session.
Han et al., (2011): Primate Cortex
explored opsins, found ArchT was 3* more light sensitive than Arch. Monkeys were given occasional juice rewards to senure alertness, but no tasks or rewards were given during recording. ArchT was expressed well in macaque cortical tissue 1 month after viral injection. 7/74 neurones did not show reductions in firing rate, spiking activity increased instead. Increased spike with a latency of 280ms after light onset, significantly longer than the latency for initiating optical quieting of the primary population of neurones. Could be a secondary homeostatic compensation for ArchT administration. Alternatively, ArchT could have unmasked other cortical projections that result in excitation or silencing of cortical neurones.
Give the explanation for the abnormal response seen in Han et al., (2011).
The secondary opponent response arises with a longer latency than the primary response. This finding is suggestive of the possibility that specific circuit elements, such as inhibitory interneurons, are playing a role in the balancing of total cortical neural activity upon pyramidal cell activity perturbation. That is, the silencing of pyramidal neurons decreases the drive to inhibitory neurons, thus increasing activity in cortical cells targeted by those inhibitory neurons; conversely, exciting pyramidal neurons increases the drive to inhibitory neurons, decreasing activity in downstream cortical neurons.
Tan et al., (2006): Allatostatin
In Drosophila. AAVs used to express AlstR in vivo in cortical & thalamic neurones of rats, ferrets, and monkeys. It eliminates activity in in vitro, isolated spinal cords of transgenic mice expressing AlstR, inactivation reversed in minutes. Vs K+ overexpression or blocking receptors via toxins tethered to PM, these approaches offer quicker temporal regulation. In Rat Barrel Cortex, showed similar results to GABAa agonist. 0.1mM AL is sufficient for max inactivation (transgenic Cre recombinase rats). AL inactivation more complete than muscimol.Field recordings reflect both presynaptic and postsynaptic activity, not individual neurones. Live ferret, visual cortex: visual stimuli, AL abolished visually evoked activity. Saline wash 11 min after AL, activity did not begin to occur until 30 mins after saline, peak reached at 40 min after washout. AlstR in LGN of macaque, via AAV. Administration was difficult here, as AL does not cross BBB, future: small AL ligand.
Bainbridge et al., (2008): Gene Therapy
Conducted in humans w/ severe retinal dystrophy (RPE65 gene, deficiency of 11-cis retinal). AAV w/RPE65 and complementary cDNA, with the promoter. Imrpoves vision in the same mutation in Swedish Briard dogs. No clinically significant improvement in visual acuity in any of the 3 patients, but microperimetry showed improved retinal function in patient 3. Retinal sensitivity improved progressively in the right eye (study eye), no improvement in the control (left eye). Thus, the patient could see small spots of light that were 1/25 as bright as those that could be seen before treatment. Patient 3 had the least advanced retinal disease at baseline.
Guler et al., (2013): Capsaicin
TRPV1 in mice using transgenic approach, specifically in DA neurones. In accordance w/ capsaicin-induced activation, we detected an elevated number of c-Fos-immunoreactive cells within most DA targets in capsaicin-injected DAT-TRPV1 mice but not controls. This can cause cell death, but no microglial activation was seen so maybe not. Then performed whole-cell voltage clamp recordings from the midbrain slices, and treatment with the highest doses of capsaicin (10mg and 32mg) evoked the most evident responses within minutes, and the capsaicin induced locomotor response returned to control levels in 15 minutes.
Wheeler et al., (2016): Magneto
Fusion of ferritin and TRPV4. Non-invasive magnetic control. AAV used, under control of cytomegalovirus promoter for DIO approach. ohon-Beard neurones expressed this channel + a calcium sensor with fluorescence GCaMP3. The neurones are involved in escape behaviour. In contrast to magneto fish, which displayed a ten-fold increase in coiling behavior upon magnetic field exposure, there was no observable change in this behavior for either control group—uninjected WT fish or ngn1::TRPV4-p2A-ferritin fish, which bicistronically express independent, unfused TRPV4 and ferritin moieties. Used in D1R+ neurones in mice, preference shown for magnetised arm of a chamber, and controls showed no preference, Removal of magnets eliminated preference.
What is the major caveat to chemogenetic approaches?
They tend to have slower pharmacokinetics that prevent cellular activation on a physiologically relevant timescale.
Kravitz et al., (2010): Parkinsonian
In vivo, basal ganglia circuitry of Cre dependent viral expression of ChR2 in striatum of D1-Cre and D2-Cre BAC transgenic mice. Bilateral excitation of indirect pathway MSNs elicited a Parkinsonian state. Activation of direct reduced freezing and locomotor deficits. Injected ChR2-YFP. Direct pathway activation led to contraversive rotations, whereas indirect pathway activation yielded ipsiversive rotations. Thus, unilateral indirect pathway activation mimics rotational behavior induced by unilateral dopamine depletion24, consistent with the classical model of basal ganglia function. Motor deficits usually thought to be from overactive indirect, underactive direct. DBS decreases efficacy of indirect pathway, but this increased direct pathway. Used 6-OHDA lesioned mice (loss of dorsomedial striatal innervation after 1 week). Direct pathway activation completely restored motor behaviour.
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